Background/Aims: In this study, we aimed to determine the levels of malondialdehyde (MDA) in patients with inflammatory bowel disease (IBD) to investigate its contribution to tissue injury in IBD. Materials and Methods: Forty-two patients with IBD (24 cases of Crohn's disease and 18 cases of ulcerative colitis) and 38 matched healthy subjects (control group) were considered for study. MDA levels were quantified by the measurement of thiobarbituric acid reactive substances. Results: Plasma MDA levels of Crohn's disease patients were significantly higher than the control group, but not higher than the ulcerative colitis patients. Plasma MDA levels of patients with ulcerative colitis were higher than the control group but not significant. Conclusion: Increased levels of plasma MDA in IBD is an important indication of oxidative stress. Patients with Crohn's disease are more susceptible to oxidative stress than patients with ulcerative colitis.

Inflammatory bowel disease (IBD), Crohn's disease and ulcerative colitis are debilitating illnesses of the bowel characterized by chronic inflammation of unknown etiology.[1] While the etiology is unknown, three main factors are assumed to play a role in the formation and progression of these diseases: (1) immune system dysfunction, (2) oxidant stress and (3) poor diet. Oxidant stress could be a major factor in IBD. Often the first attack is preceded by a stressful period in the life of the patient and flare-ups are often precipitated by emotional or physical stress. Emotional or physical stress can result in oxidative stress.[2] The state of oxidative stress may exist when there is an imbalance between the levels of antioxidant and reactive oxygen species (ROS).

Oxygen-free radicals and lipid peroxides (oxidative stress) are highly reactive and damaging compounds.[3] Oxidative stress could be a major contributing factor to the tissue injury and fibrosis that characterize IBD. It is present in IBD as it is evident by the increased production of ROS, decreased antioxidant levels (e.g., β -carotene and vitamins C and E) and decreased antioxidant enzymes in the intestinal mucosa.[4],[5],[6],[7],[8],[9]

In most of the recent studies, it has been demonstrated that inflammatory lesions of gut mucosa and smooth muscle cells have more accumulation of granulocytes than other layers.[10],[11],[12],[13],[14],[15] Those activated cells release many inflammatory mediators such as toxic oxygen metabolites, lysosomal enzymes and arachidonic acid metabolites.[15],[16],[17],[18],[19],[20],[21] In turn, superoxide anion radicals ( O 2 ), hydrogen peroxide (H 2 O 2 ) and hydroxyl radicals ( OH), secreted by neutrophils and other phagocytes accumulate within inflammatory lesions, thereby causing the impairment of the cellular membrane and cell death by leading lipid peroxidation.[22]

The previous studies have contradictory results in reporting the levels of oxidative stress in IBD patients. Several studies have shown no difference in the levels of oxidative stress between IBD patients and normal subjects.[13],[14] Other studies have shown a significant difference in the levels of oxidative stress between the IBD patients and the normal group. In this study, we therefore wanted to explore the exact levels of oxidative stress by measuring MDA - the break-down product of lipid peroxides - in the plasma of IBD patients.

Materials and Methods

Study population

Our study group comprised 42 patients (nonsmokers) with severe IBD (24 cases of Crohn's disease and 18 cases of ulcerative colitis), who presented at the gastrointestinal unit of King Khalid University Hospital (KKUH), Riyadh, Saudi Arabia and 38 healthy subjects (control group) with a mean age of 29.5±5.7 years and a body mass index (BMI) of 24.5±3.4 and who were not on medications. [Table - 1] shows the demographic characteristics of the patients with Crohn's disease and ulcerative colitis, who were under study. IBD patients were not on medications that could affect the oxidant activity. Blood samples were collected in the outpatient clinic and immediately transferred to the laboratory for the test of lipid peroxides. The study was approved by the Ethics Committee in our institute and an informed consent was obtained from all the participants.

Lipid peroxide assay

Lipid peroxides were estimated by an improved analysis of thiobarbituric acid reactive substances (TBARSs), which primarily reflects malondialdehyde (MDA), in human fluids as described previously.[23] In the presence of heat and acid, MDA reacts with thiobarbituric acid (TBA) to form a red pigment with a peak wavelength (lmax ) of 530nm. Reagents were obtained from Sigma Chemical (St. Louis, MO). Tetramethoxypropane was used to generate MDA to obtain the standard curve in the range from 0.1mM to 5.0 mM. Blood serum (200mL) was mixed with 0.2 M phosphoric acid (200 mL), 5 mM butylated hydroxytoluene (25mL) and 0.11M TBA (25mL). The mixture was incubated at 90°C for 45min and then placed on ice for a very short duration in order to stop the reaction. MDA was extracted by the addition of n-butanol (500mL). The absorptions at 530 nm and 570nm were measured using a 96-well plate reader (Specta Microplate Autoreader, Tecan, Research Triangle Park, NC).

Statistical analysis

Data are expressed as mean ± standard error of the mean (SEM) values. Comparisons between the control subjects, Crohn's disease and ulcerative colitis patients were made using ANOVA and results were considered to be statistically significant when P was < 0.05. We used post hoc test (Bonferroni test) to detect significance. Standard statistical software (SPSS, version 12; Chicago, IL) was used for the analyses.

Results

[Table - 1] shows the demographic characteristics of the studied subjects. There was no statistical difference either in age or BMI between the three study groups.

In recent years, considerable attention has been given to the role of reactive oxygen metabolites in the pathogenesis of IBD.[9],[24],[25],[26],[27],[28] Lipid peroxides and oxygen radicals are responsible for many of the damaging reactions in the cell.[3] TBARS primarily reflect malondialdehyde, a breakdown product of lipid peroxides[23] and are commonly used as a measure of oxidative stress. Walsh et al. have previously shown that TBARS highly correlate with 8-isoprostane and thus it accurately reflects lipid peroxides.[29] MDA can affect the membrane proteins by cross-linkage, rendering them useless as receptors or enzymes.[30]

Under normal physiological conditions, dietary and enzymatic antioxidants protect tissues from the damaging effects of ROS. Studies have indicated that an imbalance between the increased ROS and the decreased antioxidant defenses occurs in IBD patients.[4],[5] Decreased blood levels of vitamin C and E and decreased intestinal mucosal levels of CuZn superoxide dismutase, glutathione, vitamin A, C and E and β -carotene were reported for Crohn's disease patients.[6],[7],[8],[9],[31],[32],[33]

Studies reporting serum lipid peroxidation in IBD patients have shown contradictory results. Durak et al. have reported that tissue MDA levels in ulcerative colitis patients was significantly low indicating that the mucosa was not under oxidative stress and the defense mechanism was not impaired.[34] Tuzun et al. have demonstrated that plasma MDA levels were not different between IBD patients and control group, which was consistent with the result obtained by Bhaskar.[13],[35] In parallel with these different studies, we did not find any difference in the MDA levels in the intestinal smooth muscle cells isolated from Crohn's disease patients.[14] In contrast, increased plasma lipid peroxides in Crohn's disease patients, as estimated by TBARS concentrations, were previously reported.[36] Many other studies showed that IBD has high levels of oxidative stress and low levels of antioxidant defense,[37],[38],[39] which is consistent with this study. The significant production of MDA in Crohn's disease patients suggests that oxidative stress is a distinctive marker of this disease. On the other hand, plasma MDA levels of ulcerative colitis patients also were higher than normal but were not significantly different. However, it may be due to the inadequate number of patients with ulcerative colitis.

In conclusion, our finding of increased levels of plasma MDA in Crohn's disease group supports the increase of free radical levels in these patients. Oxidant stress may represent a distinctive characteristic of Crohn's disease. On the other hand, there was no significant difference in the plasma MDA levels between ulcerative colitis patients and control subjects. This finding is contrary to several published studies that showed higher levels of oxidative stress in IBD patients and it may be explained by experimental differences in the severity of IBD. Further investigations are required to elucidate the relationship of these changes with the pathogenesis of inflammatory bowel disease.

Acknowledgments

This work was supported by College of Medicine Research Center (CMRC) Grant (03-466) at King Saud University.